In the first step in insulin action, insulin binds to its receptor on the surface of the target cell. The insulin receptor is a transmembrane protein that possesses tyrosine-specific protein kinase activity. When insulin binds to the extracellular domain of the receptor, this activates the receptor tyrosine kinase activity. A growing body of evidence suggests that the activation of the receptor's tyrosine kinase is a necessary step in initiating the biological actions of insulin. Accordingly, we have embarked upon a search for intracellular proteins that are substrates for phosphorylation by the receptor-associated tyrosine kinase. We have identified one such substrate in rat liver plasma membranes: a glycoprotein with an apparent molecular weight of 120,000 (pp 120). In addition to being a substrate for the insulin receptor, pp120 can be phosphorylated by the receptors for epidermal growth factor and insulin-like growth factor I. pp 120 is present in liver from several species, but has not been identified in other tissues. The glycoprotein (pp120) was immunoaffinity- purified using monoclonal antibody HA4. Based on partial amino acid sequence data, pp120 has been tentatively identified as ectoATPase - an enzyme associated with hepatocyte plasma membranes. We have cloned the rat gene encoding pp120/ectoATPase. The gene contains 9 exons, and spans approximately 15 kilobase pairs of DNA. Exon 7 undergoes variable splicing. The transcript lacking exon 7 encodes an isoform in which the cytosolic domain is truncated to only 10 amino acids. The truncation deletes all three putative phosphorylation sites. When the cDNA encoding the full length isoform is expressed by transfection in 3T3 cells, it is capable of being phosphorylated by the insulin receptor tyrosine kinase. In addition, we have cloned the cDNA encoding rat pp 120/ecto-ATPase and have expressed the cDNA by transfection into cultured cells. This will enable studies of the effects of phosphorylation upon the function of the protein.